Neurochemistry and Genetics of Drosophila CaMKII

描述(由申请人提供)：局部调节突触蛋白的水平和活性对可塑性至关重要。在这个建议中，我们研究了翻译在调节CaMKII水平中的作用，并表征了EAG和dCASK与CaMKII相互作用的功能角色。果蝇身上可用的遗传工具使我们能够将我们的生化研究结果应用于设计理解行为的新策略。具体目标1.CaMKII的翻译调控和可塑性。CaMKII的新合成对长期记忆很重要，而且大部分新合成都定位于突触区域。定位和调控合成发生的机制才刚刚开始阐明，RASI/siRISC途径(FOLS)和CPEBP途径(啮齿动物和我们的初步结果)都已被涉及。在这个目标中，我们将确定哪些过程调节CaMKII的合成，以及它们与行为的关系。具体目标#2.EAG在可塑性中的非经络作用。EAG是一种电压门控性钾通道，但在果蝇中，该通道的突变体影响多个电流。我们已经确定了EAG基因座的另一个产物，它是对多个信号转导通路的激活做出反应而产生的，它编码一种具有核信号转导潜力的非通道蛋白。我们将确定这种蛋白质是否直接作用于突触和/或它是否调节基因表达。特定目的#3.dCASK调节CaMKII自磷酸化的行为效应。DCASK调节CaMKII的自磷酸化。当突触活性较低时，通过增加T306的磷酸化(阻断CaM结合)，该蛋白可以下调不活跃突触的CaMKII激活池。T306的磷酸化对随后的T287的磷酸化也有影响，T287是CaMKII上的“记忆”位点：阻止转换到激活状态。因此，dCASK是钙依赖性和非钙依赖性CaMKII活性的“增益控制器”。为此，我们将使用dCASK来调节体内CaMKII的自磷酸化水平，并评估学习和记忆的形成。这些研究将为突触过程的局部和遗传调控提供洞察力。理解这些过程是理解行为的基础。

DESCRIPTION (provided by applicant): Local regulation of the levels and activity of synaptic proteins is critical to plasticity. In this proposal, we investigate the role of translation in regulation of the levels of CaMKII and characterize the functional roles of the interaction of Eag and dCASK with CaMKII. The genetic tools available in Drosophila allow us to apply the results of our biochemical studies to the design of novel strategies for understanding behavior. Specific Aim #1. Translational control of CaMKII and plasticity. New synthesis of CaMKII is important for long-term memory, and much of the new synthesis is localized to synaptic regions. The mechanisms by which localization and regulated synthesis occurs are just beginning to be elucidated and both the rasi/siRISC pathway (flies) and CPEBP pathway (rodents and our preliminary results) have been implicated. In this aim we will determine what processes regulate CaMKII synthesis and how they relate to behavior. Specific Aim #2. Non-channel role of Eag in plasticity. Eag is a voltage-gated potassium channel, but in Drosophila, mutants in this channel affect multiple currents. We have identified an alternative product of the Eag locus that is produced in response to activation of multiple signal transduction pathways and encodes a non-channel protein with potential for nuclear signaling. We will determine if this protein functions directly at synapses and/or whether it regulates gene expression. Specific Aim #3. Behavioral effects of dCASK regulation of CaMKII autophosphorylation. dCASK regulates the autophosphorylation of CaMKII. By increasing T306 phosphorylation (which blocks CaM binding) when synaptic activity is low, this protein can downregulate the activatable pool of CaMKII at synapses that are inactive. T306 phosphorylation also has an impact on subsequent phosphorylation of T287, the 'memory' site on CaMKII: blocking switching into the active state. dCASK is therefore a 'gain controller' for both the calcium-dependent and calcium-independent activities of CaMKII. In this aim we will use dCASK to modulate the levels of CaMKII autophosphorylation in vivo and assess learning and memory formation. These studies will provide insight into local and genetic regulation of synaptic processes. Understanding these processes is fundamental to understanding behavior.